A gas atmosphere different from air atmosphere is required in the culture of biological samples such as tissues and cells conducted in the fields of the research or industry of biology, generation and biotechnology. Generally in the culture of cells, the cells in the culture fluid must be maintained at an appropriate temperature, and the culture fluid must be maintained at a constant pH as well. In the case of a culture fluid with the generally used pH buffer system of carbon dioxide—sodium hydrogen carbonate, carbon dioxide is required to be in the concentration of about 5% by volume in order to maintain pH 7.4.
In addition, the low oxygen culture of cells has recently attracted attention in many research fields. Specifically, the effects of deriving genes such as hypoxia inducible factor (HIF) in connection with biochemical reactions similar to in vivo reactions or angiogenesis and promoting the proliferation and differentiation of cells have been confirmed by the cell culture conducted under hypoxic atmosphere similar to that of a living body. Also, in the ischemia reperfusion experiment models which reproduce organ dysfunction due to blood stagnation, intracellular mechanisms have been studied in detail by exposing the cells or tissues under an anoxic condition for a certain period. Furthermore, in the drug sensitivity test with use of human cells, drug metabolism tests have been carried out by producing reducing atmosphere under an anoxic condition.
In the cultures of the biological samples described above, a given gas atmosphere is produced with a carbon dioxide incubator or a multigas incubator (referred to hereinafter as “carbon dioxide gas incubators”) and cell culture is conducted in a breathable culture vessel held in the carbon dioxide gas incubators. In this connection, multiwell plates, flasks, petri dishes, chamber slides, culture bags and the like are generally used as the culture vessel.
The parallel treatment of plural samples always involves some risk of the contamination (cross contamination) of the different sample. Coexistence of many samples within carbon dioxide gas incubators is apprehensive for the increased risk of cross contamination. Thus, carbon dioxide gas incubators having plural sample chambers have been developed, but installation of these incubators is limited due to their expensiveness. Moreover, introduction of the carbon dioxide gas incubators may be limited because of the problem with the management of high pressure gases.
For the reasons described above, a method for forming a certain gas atmosphere by using an airtight container and an atmosphere control agent to be injected into the container is also used in place of the carbon dioxide gas incubators. So far, atmosphere control agents comprising water and sodium borohydride as the main ingredients had been used in many cases of anaerobic culture and microaerophilic culture with an atmosphere control agent. The main current of the atmosphere control agent has been transferred into the products containing ascorbic acid as the main ingredient from the viewpoint of safety and reliability.
The oxygen absorbent containing the ascorbic acid components as the main ingredient can be prepared, for example, by mixing an ascorbic acid component, a metal salt, activated carbon and water as disclosed in Japanese Patent Laid-Open Publication No. H10-314581 (lit. 1). In addition, it is possible to control not only the amount of carbon dioxide to be generated but also the concentration of carbon dioxide in a container having a fixed volume by adding an alkaline earth metal hydroxide to the oxygen absorbent as disclosed in Japanese Patent Laid-Open Publication No. H10-327845 (lit. 2). Furthermore, Japanese Patent Laid-Open Publication No. H09-252766 (lit. 3) discloses that an atmosphere control agent which may form the appropriate concentrations of oxygen and carbon dioxide depending on the biological materials has been developed and used.
In the field of conserving foods with an atmosphere control agent, it is known that acetaldehyde generates with the deterioration of foods or by the oxidation of ethanol in the use of an iron type oxygen scavenger in combination with ethanol, and an oxygen scavenger having the acetaldehyde-removing ability has been developed in order to remove acetaldehyde (Japanese Patent Laid-Open Publication No. S62-40273 (lit. 4). However, when no aldehyde generates from an object for preservation and an atmosphere control agent and ethanol are not used in combination, it has not been known that aldehyde will generate with the use of an atmosphere control agent. Furthermore, in the field of cell culture with an atmosphere control agent, no examination has been done on the effect of aldehyde on the cell culture so far as the present inventors know.
The present applicant has hitherto proposed in the field of the cell culture a method for adjusting the atmosphere of a low oxygen concentration as described, for example, in Japanese Patent Laid-Open Publication No. H09-0252766.